Purification and biochemical characterization of recombinant hirudin produced by Saccharomyces cerevisiae

Abstract

Recombinant hirudin was produced by the yeast Saccharomyces cerevisiae using the alpha-pheromone prepro sequence to direct its secretion into the culture medium. The secreted hirudin was isolated to greater than or equal to 95% purity as measured by 205-nm absorbance integration from a reverse-phase chromatogram. One major activity peak corresponding to the complete, correctly processed molecule and two minor activity peaks corresponding to C-terminally truncated forms were identified. The primary structure of the major peak, determined by N-terminal sequencing of tryptic peptides, was that predicted from the cDNA sequence, and the molecular mass analyzed by fast atom bombardment mass spectrometry (FAB-MS) was 6892.6 (calculated 6892.5). UV spectral analysis suggested that, in contrast to the natural molecule, recombinant hirudin produced by S. cerevisiae is not sulfated.

C-terminal processing of barley alpha-amylase 1 in malt, aleurone protoplasts, and yeast.

“…Accordingly, processing of AMY1-3 and AMY14 with a penultimate arginine is efficiently catalyzed by malt carboxypeptidase II. C-terminal processing ofheterologous proteins secreted from yeast has previously been found for human atrial natriuretic peptide (16), human epidermal growth factor (17), and leech hirudin HV2 (18). Two of these (16,17) possess a basic residue at either the penultimate or the terminal position.…”

“…Apparently a carboxypeptidase specific for basic residues operates in the secretory pathway, and a likely candidate is the KEXI-encoded membrane-bound carboxypeptidase known to remove C-terminal Lys-Arg from a-factor and killer toxin, secreted by certain yeast strains (19). Leech hirudin, however, has no basic amino acid near the C terminus, and its processing, which progressed with culture time, is more likely due to vacuolar carboxypeptidases released by cell lysis (18). Using the kexi yeast mutant M204-8C, we have Kinetic parameters toward p-nitrophenyl maltoheptaoside were determined at 37TC in a coupled assay using yeast maltase at pH 6 (Fig.…”

Characterization, stability and refolding of recombinant hirudin

“…Upon determination of the complete primary structure of natural hirudin variants Harvey et al, 1986;Tripier, 1988;Scharf et al, 1989), the protein has now become available from recombinant sources in larger quantities Fortkamp et al, 1986;Harvey et al, 1986;Dodt et al, 1986;Braun et al, 1988;Crause et al, 1988;Riehl-Bellon et al, 1989) allowing a physicochemical characterization of the inhibitor purified to homogeneity. The observed ultraviolet absorption, dichroic absorption, and fluorescence emission spectra of recombinant hirudin correspond to results on its solution and crystal structure, as obtained by 2D-NMR (Folkers et al, 1989;Haruyama and Wuthrich, 1989) and X-ray crystallography (Rydel et al, 1990).…”

Heterologous Expression of Human Cholecystokinin in Saccharomyces cerevisiae

“…Analysis of CCK-Gly secreted from yeast transformants by both anion exchange chromatography and mass spectrometry revealed only the non-sulfated forms. The inability of S. cerevisiae to sulfate tyrosine residues was also observed for the leech anticoagulant, hirudin, which requires tyrosyl sulfation for full activity (Riehl-Bellon et al, 1989).…”